Novel aminopyridine derivatives having aurora a selective inhibitory action

ABSTRACT

The present invention relates to a compound of Formula (I): wherein: n is 0 or 1; X is O or CH 2 ; R 1  is H or C 1-2  alkyl; R 2  is H or C 1-3  alkyl; R 3  and R 4  are each independently H or C 1-2  alkyl, where the alkyl may be substituted with one to three of the same or different substituents selected from R 10  R 5  is H or OCH 3 ; R 10  is F or Cl; or a pharmaceutically acceptable salt or ester thereof.

TECHNICAL FIELD

The present invention relates to novel aminopyridine derivatives whichare useful in the pharmaceutical field, and more particularly, to thosewhich inhibit the growth of tumor cells based on an Aurora A selectiveinhibitory action and exhibit an antitumor effect, and also to an AuroraA selective inhibitor and an antitumor agent containing them.

BACKGROUND ART

Aurora kinase is a serine/threonine kinase involved in cell division.With regard to the Aurora kinase, three subtypes of A, B and C are knownat present, and they have very high homology to each other. Aurora Aparticipates in the maturation and distribution of centrosome or in theformation of spindle body. On the other hand, it is believed that AuroraB participates in the aggregation and pairing of chromosome, a spindlecheckpoint and cytoplasm division [Nat. Rev. Mol. Cell Biol., No. 4, pp.842-854]. Also, it is believed that Aurora C acts similarly as a resultof interaction with Aurora B [J. Biol. Chem., Epub ahead (2004)]. Fromthe fact that high expression of Aurora A has been hitherto confirmed inmany cancer cells; that high expression of Aurora A in normal cellsleads to transformation of normal cell strains of rodent; and the like,Aurora A, being one of oncogenes, is recognized to be an adequate targetfor an antitumor agent [EMBO J., No. 17, pp. 3052-3065 (1998)].

There is another report that cancer cells in which Aurora A is highlyexpressed have a resistance to paclitaxel [Cancer Cell, Vol. 3, pp.51-62 (2003)]. Meanwhile, with regard to the Aurora kinase inhibitor,development of subtype-selective drugs has been thought to be difficultin view of high homology among subtypes, protein structure analysis andthe like; and although there have been known reports on drugs such asZM447439 which inhibit both Aurora A and Aurora B at the same time [J.Cell Biol., No. 161, pp. 267-280 (2003); J. Cell Biol., No. 161, pp.281-294, (2003); Nat. Med., No. 10, pp. 262-267, (2004)], no reportconcerning Aurora A selective drugs have been known. Thus, in thosereports, disclosed is the antitumor effect only for the case where adrug which inhibits both Aurora A and Aurora B at the same time issolely administered. In addition, there has been also reported a resultthat in a drug which inhibits both Aurora A and Aurora B at the sametime, the Aurora kinase inhibiting action attenuates the action ofpaclitaxel [J. Cell Biol., No. 161, pp. 281-294, (2003)].

On the other hand, patent applications concerning compounds having anAurora kinase inhibiting action have been previously filed(WO2002/022606, WO2002/022602, WO2002/0220601, W02006/046734).

DISCLOSURE OF THE INVENTION

The purpose of the present invention is to provide novel aminopyridinederivatives which show an excellent Aurora A selective inhibitory actionand cell-growth inhibitory action based on the foregoing, therebyachieving a synergistic action by a combined use with other antitumoragent(s).

The present inventors have synthesized a variety of novel aminopyridinederivatives and found that the compound represented by the followingFormula (I) shows an excellent Aurora A selective inhibitory action.

Thus, the invention relates to a compound of Formula (I):

wherein:

n is 0 or 1;

X is O or CH₂;

R¹ is H or C₁₋₂ alkyl;

R² is H or C₁₋₃ alkyl;

R³ and R⁴ are each independently H or C₁₋₂ alkyl, where the alkyl may besubstituted with one to three of the same or different substituentsselected from R¹⁰;

R⁵ is H or OCH₃;

R¹⁰ is F or Cl;

or a pharmaceutically acceptable salt or ester thereof.

The invention also relates to a combined preparation for simultaneous,separate or sequential administration in the treatment of cancer,comprising two separate preparations which are:

(i) a preparation comprising, together with a pharmaceuticallyacceptable carrier or diluent, a compound represented by theabove-described Formula (I) or a pharmaceutically acceptable salt orester thereof; and

(ii) a preparation comprising, together with a pharmaceuticallyacceptable carrier or diluent, one antitumor agent selected from thegroup consisting of antitumor alkylating agents, antitumorantimetabolites, antitumor antibiotics, plant-derived antitumor agents,antitumor platinum coordination compounds, antitumor camptothecinderivatives, antitumor tyrosine kinase inhibitors, monoclonalantibodies, interferons, biological response modifiers and otherantitumor agents as well as pharmaceutically acceptable salt(s) orester(s) thereof, wherein:

the antitumor alkylating agent is nitrogen mustard N-oxide,cyclophosphamide, ifosfamide, melphalan, busulfan, mitobronitol,carboquone, thiotepa, ranimustine, nimustine, temozolomide or carmustin;

the antitumor antimetabolite is methotrexate, 6-mercaptopurine riboside,mercaptopurine, 5-fluorouracil, tegafur, doxyfluridine, carmofur,cytarabine, cytarabine ocfosfate, enocitabine, S-1, gemcitabine,fludarabine or pemetrexed disodium;

the antitumor antibiotic is actinomycin D, doxorubicin, daunorubicin,neocarzinostatin, bleomycin, peplomycine, mitomycin C, aclarubicin,pirarubicin, epirubicin, zinostatin stimalamer, idarubicin, sirolimus orvalrubicin;

the plant-derived antitumor agent is vincristine, vinblastine,vindesine, etoposide, sobuzoxane, docetaxel, paclitaxel or vinorelbine;

the antitumor platinum coordination compound is cisplatin, carboplatin,nedaplatin or oxaliplatin;

the antitumor camptothecin derivative is irinotecan, topotecan oreamptothecin;

the antitumor tyrosine kinase inhibitor is gefitinib, imatinib,sorafenib, sunitinib, dasatinib,or erlotinib;

the monoclonal antibody is cetuximab, rituximab, bevacizumab,alerntuzumab or trastuzumab;

the interferon is interferon α, interferon α-2a, interferon α-2b,interferon β, interferon γ-1a or interferon γ-n1;

the biological response modifier is krestin, lentinan, sizofiran,picibanil or ubenimex; and

the other antitumor agent is mitoxantrone, L-asparaginase, procarbazine,dacarbazine, hydroxycarbamide, pentostatin, tretinoin, alefacept,darbepoetin alfa, anastrozole, exemestane, bicalutamide, leuprolelin,flutamide, fulvestrant, pegaptanib octasodium, denileukin diftitox,aldesleukin, thyrotropin alfa, arsenic trioxide, bortezomib,capecitabine or goserelin.

The invention further relates to a pharmaceutical compositioncomprising, together with a pharmaceutically acceptable carrier ordiluent, a compound represented by the above-described Formula (I) or apharmaceutically acceptable salt or ester thereof, and an antitumoragent selected from the group consisting of antitumor alkylating agents,antitumor antimetabolites, antitumor antibiotics, plant-derivedantitumor agents, antitumor platinum coordination compounds, antitumorcamptothecin derivatives, antitumor tyrosine kinase inhibitors,monoclonal antibodies, biological response modifiers and other antitumoragents (here, the definition of each antitumor agent is the same as thatdefined hereinabove) or a pharmaceutically acceptable salt or esterthereof.

The invention still further relates to a method for the treatment ofcancer, comprising administering simultaneously, separately orsequentially a therapeutically effective amount of a compoundrepresented by the above-described Formula (I) or a pharmaceuticallyacceptable salt or ester thereof in combination with a therapeuticallyeffective amount of an antitumor agent selected from the groupconsisting of antitumor alkylating agents, antitumor antimetabolites,antitumor antibiotics, plant-derived antitumor agents, antitumorplatinum coordination compounds, antitumor camptothecin derivates,antitumor tyrosine kinase inhibitors, monoclonal antibodies,interferons, biological response modifiers and other antitumor agents(here, definition of each antitumor agent is the same as that definedhereinabove) or a pharmaceutically acceptable salt or ester thereof.

Furthermore, the invention relates to the use of an Aurora selective Ainhibitor for the manufacture of a medicament for the treatment ofcancer; and the use of an Aurora selective A inhibitor in combinationwith an antitumor agent for the manufacture of a medicament for thetreatment of cancer; and also relates to a method of treating cancer toa mammal (particularly a human) which comprises administering to saidmammal a therapeutically effective amount of an Aurora selective Ainhibitor; and a method of treating cancer in a mammal (particularly ahuman) which comprises administering to said mammal a therapeuticallyeffective amount of an Aurora selective A inhibitor in combination witha therapeutically effective amount of an antitumor agent.

The invention relates to a pharmaceutical composition comprising asactive ingredient an Aurora selective A inhibitor; and a pharmaceuticalcomposition comprising as active ingredient an Aurora selective Ainhibitor, together with an antitumor agent.

Next, symbols and terms used in the present specification will beexplained.

The term “alkyl” is intended to include both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. For example, C₁₋₆, as in the term “C₁₋₆ alkyl” is definedto include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear orbranched arrangement. For example, the term “C₁₋₆ alkyl” specificallyincludes methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl,pentyl, hexyl, and so on. Generally, the term “C_(m-n) alkyl” is definedto include groups having m to n carbons in a linear or branchedarrangement, where m and n each independently are an integer of 1 to 6but n is greater than m.

The term “selective inhibitor of Aurora A” used in the presentspecification is a compound or a drug which selectively inhibits AuroraA as compared with Aurora B. The “selective inhibitor of Aurora A” ispreferably a compound or a drug of which inhibitory activities againstAurora A are at least ten times the activities against Aurora B; andmore preferably a compound or a drug of which inhibitory activitiesagainst Aurora A are at least hundred times the activities againstAurora B.

Explanation for the term “pharmaceutically acceptable salt of esterthereof” or the term “pharmaceutically acceptable carrier or diluent”used in the specification still will be given later.

The term “treatment of cancer” as used in the specification meansinhibition of cancer cell growth by administering an antitumor agent toa cancer patient. Preferably, this treatment enables retrogression ofcancer growth, that is, reduction in the measurable cancer size. Morepreferably, such treatment completely eliminates cancer.

The term “cancer” as used in the specification refers to solid cancerand hematopoietic cancer. Here, examples of solid cancer includecerebral tumor, head and neck cancer, esophageal cancer, thyroid cancer,small cell lung cancer, non-small cell lung cancer, breast cancer,stomach cancer, gallbladder and bile duct cancer, liver cancer, pancreascancer, colon cancer, rectal cancer, ovarian cancer, chorioepithelioma,uterine cancer, cervical cancer, renal pelvic and ureteral cancer,bladder cancer, prostate cancer, penile cancer, testicular cancer,embryonal cancer, wilms tumor, skin cancer, malignant melanoma,neuroblastoma, osteosarcoma, Ewing's tumor and soft tissue sarcoma. Onthe other hand, examples of hematopoietic cancer include acute leukemia,chronic lymphatic leukemia, chronic myelocytic leukemia, polycythemiavera, malignant lymphoma, multiple myeloma and non-Hodgkins' lymphoma.

The term “preparation” as used in the specification includes oralpreparations and parenteral preparations. Examples of oral preparationsinclude tablets, capsules, powders and granules, while examples ofparenteral preparations include sterilized liquid preparations such assolutions or suspensions, specifically injections or drip infusions.Preferably, they are intravenous injections or intravenous dripinfusions, and more preferably intravenous drip infusions.

The term “combined preparation” as used in the specification refers tothose comprising two or more preparations for simultaneous, separate orsequential administration in the treatment, and such preparation may bea so-called kit type preparation or pharmaceutical composition. The term“combined preparation” also includes those having one or morepreparations further combined with the combined preparation comprisingtwo separate preparations used in the treatment of cancer.

The two separate preparations described above can be further combinedwith, in combination with a pharmaceutically acceptable carrier ordiluent, at least one preparation comprising at least one antitumoragent selected from the group consisting of antitumor alkylating agents,antitumor antimetabolites, antitumor antibiotics, plant-derivedantitumor agents, antitumor platinum coordination compounds, antitumorcamptothecin derivatives, antitumor tyrosine kinase inhibitors,monoclonal antibodies, interferons, biological response modifiers andother antitumor agents (here, definition of each antitumor agent is thesame as that defined above), or a pharmaceutically acceptable salt orester thereof. In this case, the above-mentioned at least onepreparation that has been further combined can be administeredsimultaneously, separately or sequentially with respect to the twoseparate preparations. For example, a combined preparation comprisingthree preparations may include that is comprised of a preparationincluding a preparation containing the compound represented by the aboveFormula (I), a preparation containing 5-fluorouracil and a preparationcontaining leucovorin.

Here, in the above-mentioned combined preparation, either or both of thetwo separate preparations may be an oral preparation; and also one maybe an oral preparation, while another may be a parental preparation(injections or drip infusions).

The term “preparation” according to the invention may usually comprise atherapeutically effective amount of a compound according to theinvention, together with a pharmaceutically acceptable carrier ordiluent. This technique of formulation is considered to be a technicalcommon knowledge to those having ordinary skill in the pertinent art andis well known. Preferably, oral preparations, intravenous drip infusionsor injections can be prepared in combination with a pharmaceuticallyacceptable carrier or diluent, by various methods that are well known inthe art.

In the case of using the combined preparation according to theinvention, the term “administration” as used in the presentspecification refers to parenteral administration and/or oraladministration, and preferably oral administration. Thus, when acombined preparation is administered, both administrations may beparenteral; one administration may be parenteral while the other may beoral; or both administrations may be oral. Preferably, both preparationsin the combined preparation are administered orally. Here, the term“parenteral administration” is, for example, intravenous administration,subcutaneous administration or intramuscular administration, andpreferably it is intravenous administration. Even when three or morepreparations are combined and administered, every preparation may beorally administered.

In the embodiment of the present invention, a compound represented bythe above Formula (I) may be administered simultaneously with otherantitumor agent(s). Further, it is possible to administer the compoundrepresented by the above Formula (I) first and then another antitumoragent consecutively, or alternatively it is possible to administeranother antitumor agent first and then the compound represented by theabove Formula (I) consecutively. It is also possible to administer thecompound represented by the above Formula (I) first and then separatelyadminister another antitumor agent after a while, or alternatively it ispossible to administer another antitumor agent first and then separatelyadminister the compound represented by the above Formula (I) after awhile. The order and the time interval for the administration may beappropriately selected by a person skilled in the art in accordancewith, for example, a preparation containing the compound represented bythe above Formula (I) used and a preparation containing an antitumoragent that is used in combination therewith, the type of the cancercells to be treated and the condition of the patient. For example, inthe case of administering the compound represented by the above Formula(I) and paclitaxel or docetaxel, preferably paclitaxel or docetaxel isadministered first, and then the compound represented by the aboveFormula (I) is administered sequentially or separately after a while.

The term “simultaneously” as used in the specification refers to the useof preparations for the treatment substantially at the same time,whereas the term “separately” refers to the separate use of preparationsfor the treatment at different times such that, for example, one agentis used on the first day and another agent is used on the second day forthe treatment. The term “sequentially” refers to the use of preparationsin such an order that, for example, one agent is first used and anotheragent is used after a predetermined period of time for the treatment.

The term “antitumor alkylating agent” as used in the presentspecification refers to an alkylating agent having antitumor activity,and the term “alkylating agent” herein generally refers to an agentgiving an alkyl group in the alkylation reaction in which a hydrogenatom of an organic compound is substituted with an alkyl group. The term“antitumor alkylating agent” may be exemplified by nitrogen mustardN-oxide, cyclophosphamide, ifosfamide, melphalan, busulfan,mitobronitol, carboquone, thiotepa, ranimustine, nimustine, temozolomideor carmustine.

The term “antitumor antimetabolite” as used in the specification refersto an antimetabolite having antitumor activity, and the term“antimetabolite” herein includes, in a broad sense, substances whichdisturb normal metabolism and substances which inhibit the electrontransfer system to prevent the production of energy-rich intermediates,due to their structural or functional similarities to metabolites thatare important for living organisms (such as vitamins, coenzymes, aminoacids and saccharides). The term “antitumor antimetabolites” may beexemplified methotrexate, 6-mercaptopurine riboside, mercaptopurine,5-fluorouracil, tegafur, doxifluridine, carmofur, cytarabine, cytarabineocfosfate, enocitabine, S-1, gemcitabine, fludarabine or pemetrexeddisodium, and preferred are 5-fluorouracil, S-1, gemcitabine and thelike.

The term “antitumor antibiotic” as used in the specification refers toan antibiotic having antitumor activity, and the “antibiotic” hereinincludes substances that are produced by microorganisms or by organicsynthesis and inhibit cell growth and other functions of microorganismsand of other living organisms. The term “antitumor antibiotic” may beexemplified by actinomycin D, doxorubicin, daunorubicin,neocarzinostatin, bleomycin, peplomycin, mitomycin C, aclarubicin,pirarubicin, epirubicin, zinostatin stimalamer, idarubicin, sirolimus orvalrubicin.

The term “plant-derived antitumor agent” as used in the specificationincludes compounds having antitumor activities which originate fromplants, or compounds prepared by applying chemical modification to theforegoing compounds. The term “plant-derived antitumor agent” may beexemplified by vincristine, vinblastine, vindesine, etoposide,sobuzoxane, docetaxel, paclitaxel and vinorelbine, and preferred anddocetaxel and paclitaxel.

The term “antitumor camptothecin derivative” as used in thespecification refers to compounds that are structurally related tocamptothecin and inhibit cancer cell growth, including camptothecin perse. The term “antitumor camptothecin derivative” is not particularlylimited to, but may be exemplified by, camptothecin,10-hydroxycamptothecin, topotecan, irinotecan or 9-aminocamptothecin,with camptothecin, topotecan and irinotecan being preferred. Further,irinotecan is metabolized in vivo and exhibits antitumor effect asSN-38. The action mechanism and the activity of the camptothecinderivatives are believed to be virtually the same as those ofcamptothecin (e.g., Nitta, et al., Gan to Kagaku Ryoho, 14, 850-857(1987)).

The term “antitumor platinum coordination (platinum-complex) compound”as used in the specification refers to a platinum coordination compoundhaving antitumor activity, and the term “platinum coordination compound”herein refers to a platinum coordination compound which providesplatinum in ion form. Preferred platinum compounds include cisplatin;cis-diamminediaquoplatinum (II)-ion; chloro(diethylenetriamine)-platinum(II) chloride; dichloro(ethylenediamine)-platinum (II);diammine(1,1-cyclobutanedicarboxylato) platinum (II) (carboplatin);spiroplatin; iproplatin; diammine(2-ethylmalonato)platinum (II);ethylenediaminemalonatoplatinum (II);aqua(1,2-diaminodicyclohexane)sulfatoplatinum (II);aqua(1,2-diaminodicyclohexane)malonatoplatinum (II);(1,2-diaminocyclohexane)malonatoplatinum (II);(4-carboxyphthalato)(1,2-diaminocyclohexane) platinum (II);(1,2-diaminocyclohexane)-(isocitrato)platinum (II);(1,2-diaminocyclohexane)oxalatoplatinum (II); ormaplatin; tetraplatin;carboplatin, nedaplatin and oxaliplatin, and preferred is carboplatin oroxaliplatin. Further, other antitumor platinum coordination compoundsmentioned in the specification are known and are commercially availableand/or producible by a person having ordinary skill in the art byconventional techniques.

The term “antitumor tyrosine kinase inhibitor” as used in thespecification refers to a tyrosine kinase inhibitor having antitumoractivity, and the term “tyrosine kinase inhibitor” herein refers to achemical substance inhibiting “tyrosine kinase” which transfers aγ-phosphate group of ATP to a hydroxy group of a specific tyrosine inprotein. The term “antitumor tyrosine kinase inhibitor” may beexemplified by gefitinib, imatinib, sorafenib, sunitinib, dasatinib, orerlotinib.

The term “monoclonal antibody” as used in the specification, which isalso known as single clonal antibody, refers to an antibody produced bya monoclonal antibody-producing cell, and examples thereof includecetuximab, bevacizumab, rituximab, alemtuzumab and trastuzumab.

The term “interferon” as used in the specification refers to aninterferon having antitumor activity, and it is a glycoprotein having amolecular weight of about 20,000 which is produced and secreted by mostanimal cells upon viral infection. It has not only the effect ofinhibiting viral growth but also various immune effector mechanismsincluding inhibition of growth of cells (in particular, tumor cells) andenhancement of the natural killer cell activity, thus being designatedas one type of cytokine. Examples of “interferon” include interferon α,interferon α-2a, interferon α-2b, interferon β, interferon γ-1a andinterferon γ-n1.

The term “biological response modifier” as used in the specification isthe so-called biological response modifier or BRM and is generally thegeneric term for substances or drugs for modifying the defensemechanisms of living organisms or biological responses such as survival,growth or differentiation of tissue cells in order to direct them to beuseful for an individual against tumor, infection or other diseases.Examples of the “biological response modifier” include krestin,lentinan, sizofiran, picibanil and ubenimex.

The term “other antitumor agent” as used in the specification refers toan antitumor agent which does not belong to any of the above-describedagents having antitumor activities. Examples of the “other antitumoragent” include mitoxantrone, L-asparaginase, procarbazine, dacarbazine,hydroxycarbamide, pentostatin, tretinoin, alefacept, darbepoetin alfa,anastrozole, exemestane, bicalutamide, leuprorelin, flutamide,fulvestrant, pegaptanib octasodium, denileukin diftitox, aldesleukin,thyrotropin alfa, arsenic trioxide, bortezomib, capecitabine, andgoserelin.

The above-described terms “antitumor alkylating agent”, “antitumorantimetabolite”, “antitumor antibiotic”, “plant-derived antitumoragent”, “antitumor platinum coordination compound”, “antitumorcamptothecin derivative”, “antitumor tyrosine kinase inhibitor”,“monoclonal antibody”, “interferon”, “biological response modifier” and“other antitumor agent” are all known and are either commerciallyavailable or producible by a person skilled in the art by methods knownper se or by well-known or conventional methods. The process forpreparation of gefitinib is described, for example, in U.S. Pat. No.5,770,599; the process for preparation of cetuximab is described, forexample, in WO 96/40210; the process for preparation of bevacizumab isdescribed, for example, in WO 94/10202; the process for preparation ofoxaliplatin is described, for example, in U.S. Pat. Nos. 5,420,319 and5,959,133; the process for preparation of gemcitabine is described, forexample, in U.S. Pat. Nos. 5,434,254 and 5,223,608; and the process forpreparation of camptothecin is described in U.S. Pat. Nos. 5,162,532,5,247,089, 5,191,082, 5,200,524, 5,243,050 and 5,321,140; the processfor preparation of irinotecan is described, for example, in U.S. Pat.No. 4,604,463; the process for preparation of topotecan is described,for example, in U.S. Pat. No. 5,734,056; the process for preparation oftemozolomide is described, for example, in JP-B No. 4-5029; and theprocess for preparation of rituximab is described, for example, in JP-WNo. 2-503143.

The above-mentioned antitumor alkylating agents are commerciallyavailable, as exemplified by the following: nitrogen mustard N-oxidefrom Mitsubishi Pharma Corp. as Nitromin (tradename); cyclophosphamidefrom Shionogi & Co., Ltd. as Endoxan (tradename); ifosfamide fromShionogi & Co., Ltd. as Ifomide (tradename); melphalan fromGlaxoSmithKline Corp. as Alkeran (tradename); busulfan from TakedaPharmaceutical Co., Ltd. as Mablin (tradename); mitobronitol from KyorinPharmaceutical Co., Ltd. as Myebrol (tradename); carboquone from SankyoCo., Ltd. as Esquinon (tradename); thiotepa from Sumitomo PharmaceuticalCo., Ltd. as Tespamin (tradename); ranimustine from Mitsubishi PharmaCorp. as Cymerin (tradename); nimustine from Sankyo Co., Ltd. as Nidran(tradename); temozolomide from Schering Corp. as Temodar (tradename);and carmustine from Guilford Pharmaceuticals Inc. as Gliadel Wafer(tradename).

The above-mentioned antitumor antimetabolites are commerciallyavailable, as exemplified by the following: methotrexate from TakedaPharmaceutical Co., Ltd. as Methotrexate (tradename); 6-mercaptopurineriboside from Aventis Corp. as Thioinosine (tradename); mercaptopurinefrom Takeda Pharmaceutical Co., Ltd. as Leukerin (tradename);5-fluorouracil from Kyowa Hakko Kogyo Co., Ltd. as 5-FU (tradename);tegafur from Taiho Pharmaceutical Co., Ltd. as Futraful (tradename);doxyfluridine from Nippon Roche Co., Ltd. as Furutulon (tradename);carmofur from Yamanouchi Pharmaceutical Co., Ltd. as Yamafur(tradename); cytarabine from Nippon Shinyaku Co., Ltd. as Cylocide(tradename); cytarabine ocfosfate from Nippon Kayaku Co., Ltd. asStrasid (tradename); enocitabine from Asahi Kasei Corp. as Sanrabin(tradename); S-1 from Taiho Pharmaceutical Co., Ltd. as TS-1(tradename); gemcitabine from Eli Lilly & Co. as Gemzar (tradename);fludarabine from Nippon Schering Co., Ltd. as Fludara (tradename); andpemetrexed disodium from Eli Lilly & Co. as Alimta (tradename).

The above-mentioned antitumor antibiotics are commercially available, asexemplified by the following: actinomycin D from Banyu PharmaceuticalCo., Ltd. as Cosmegen (tradename); doxorubicin from Kyowa Hakko KogyoCo., Ltd. as adriacin (tradename); daunorubicin from Meiji Seika KaishaLtd. as Daunomycin; neocarzinostatin from Yamanouchi Pharmaceutical Co.,Ltd. as Neocarzinostatin (tradename); bleomycin from Nippon Kayaku Co.,Ltd. as Bleo (tradename); pepromycin from Nippon Kayaku Co, Ltd. asPepro (tradename); mitomycin C from Kyowa Hakko Kogyo Co., Ltd. asMitomycin (tradename); aclarubicin from Yamanouchi Pharmaceutical Co.,Ltd. as Aclacinon (tradename); pirarubicin from Nippon Kayaku Co., Ltd.as Pinorubicin (tradename); epirubicin from Pharmacia Corp. asPharmorubicin (tradename); zinostatin stimalamer from YamanouchiPharmaceutical Co., Ltd. as Smancs (tradename); idarubicin fromPharmacia Corp. as Idamycin (tradename); sirolimus from. Wyeth Corp. asRapamune (tradename); and vairubicin from Anthra Pharmaceuticals Inc. asValstar (tradename).

The above-mentioned plant-derived antitumor agents are commerciallyavailable, as exemplified by the following: vincristine from Shionogi &Co., Ltd. as Oncovin (tradename); vinblastine from Kyorin PharmaceuticalCo., Ltd. as Vinblastine (tradename); vindesine from Shionogi & Co.,Ltd. as Fildesin (tradename); etoposide from Nippon Kayaku Co., Ltd. asLastet (tradename); sobuzoxane from Zenyaku Kogyo Co., Ltd. as Perazolin(tradename); docetaxel from Aventis Corp. as Taxsotere (tadename);paclitaxel from Bristol-Myers Squibb Co. as Taxol (tradename); andvinorelbine from Kyowa Hakko Kogyo Co., Ltd. as Navelbine (tradename).

The above-mentioned antitumor platinum coordination compounds arecommercially available, as exemplified by the following: cisplatin fromNippon Kayaku Co., Ltd. as Randa (tradename); carboplatin fromBristol-Myers Squibb Co. as Paraplatin (tradename); nedaplatin fromShionogi & Co., Ltd. as Aqupla (tradename); and oxaliplatin fromSanofi-Synthelabo Co. as Eloxatin (tradename).

The above-mentioned antitumor camptothecin derivatives are commerciallyavailable, as exemplified by the following: irinotecan from YakultHonsha Co., Ltd. as Campto (tradename); topotecan from GlaxoSmithKlineCorp. as Hycamtin (tradename); and camptothecin from Aldrich ChemicalCo., Inc., U.S.A.

The above-mentioned antitumor tyrosine kinase inhibitors arecommercially available, as exemplified by the following: gefitinib fromAstraZeneca Corp. as Iressa (tradename); imatinib from Novartis AG asGleevec (tradename); sorafenib from Bayer as Nexavar (tradename);sunitinib from Pfizer as Sutent (tradename); dasatinib from BristolMyers Squibb as Sprycel (tradename); and erlotinib from OSIPharmaceuticals Inc. as Tarceva (tradename).

The above-mentioned monoclonal antibodies are commercially available, asexemplified by the following: cetuximab from Bristol-Myers Squibb Co. asErbitux (tradename); bevacizumab from Genentech, Inc. as Avastin(tradename); rituximab from Biogen Idec Inc. as Rituxan (tradename);alemtuzumab from Berlex Inc. as Campath (tradename); and trastuzumabfrom Chugai Pharmaceutical Co., Ltd. as Herceptin (tradename).

The above-mentioned interferons are commercially available, asexemplified by the following: interferon a from Sumitomo PharmaceuticalCo., Ltd. as Sumiferon (tradename); interferon α-2a from TakedaPharmaceutical Co., Ltd. as Canferon-A (tradename); interferon α-2b fromSchering-Plough Corp. as Intron A (tradename); interferon β from MochidaPharmaceutical Co., Ltd. as IFNIβ (tradename); interferon γ-1a fromShionogi & Co., Ltd. as Imunomax-γ (tradename); and interferon γ-n1 fromOtsuka Pharmaceutical Co., Ltd. as Ogamma (tradename).

The above-mentioned biological response modifiers are commerciallyavailable, as exemplified by the following: krestin from Sankyo Co.,Ltd. as krestin (tradename); lentinan from Aventis Corp. as Lentinan(tradename); sizofiran from Kaken Seiyaku Co., Ltd. as Sonifiran(tradename); picibanil from Chugai Pharmaceutical Co., Ltd. as Picibanil(tradename); and ubenimex from Nippon Kayaku Co., Ltd. as Bestatin(tradename).

The above-mentioned other antitumor agents are commercially available,as exemplified by the following: mitoxantrone from Wyeth Lederle Japan,Ltd. as Novantrone (tradename); L-asparaginase from Kyowa Hakko KogyoCo., Ltd. as Leunase (tradename); procarbazine from Nippon Roche Co.,Ltd. as Natulan (tradename); dacarbazine from Kyowa Hakko Kogyo Co.,Ltd. as Dacarbazine (tradename); hydroxycarbamide from Bristol-MyersSquibb Co. as Hydrea (tradename); pentostatin from Kagaku Oyobi KesseiRyoho Kenkyusho as Coforin (tradename); tretinoin from Nippon Roche Co.,Ltd. As Vesanoid (tradename); alefacept from Biogen Idec Inc. as Amevive(tradename); darbepoetin alfa from Amgen Inc. as Aranesp (tradename);anastrozole from AstraZeneca Corp. as Arimidex (tradename); exemestanefrom Pfizer Inc. as Aromasin (tradename); bicalutamide from AstraZenecaCorp. as Casodex (tradename); leuprorelin from Takeda PharmaceuticalCo., Ltd. as Leuplin (tradename); flutamide from Schering-Plough Corp.as Eulexin (tradename); fulvestrant from AstraZeneca Corp. as Faslodex(tradename); pegaptanib octasodium from Gilead Sciences, Inc. as Macugen(tradename); denileukin diftitox from Ligand Pharmaceuticals Inc. asOntak (tradename); aldesleukin from Chiron Corp. as Proleukin(tradename); thyrotropin alfa from. Genzyme Corp. as Thyrogen(tradename); arsenic trioxide from Cell Therapeutics, Inc. as Trisenox(tradename); bortezomib from Millennium Pharmaceuticals, Inc. as Velcade(tradename); capecitabine from Hoffmann-La Roche, Ltd. as Xeloda(tradename); and goserelin from AstraZeneca Corp. as Zoladex(tradename).

The term “antitumor agent” as used in the specification includes theabove-described “antitumor alkylating agent”, “antitumorantimetabolite”, “antitumor antibiotic”, “plant-derived antitumoragent”, “antitumor platinum coordination compound”, “antitumorcamptothecin derivative”, “antitumor tyrosine kinase inhibitor”,“monoclonal antibody”, “interferon”, “biological response modifier” and“other antitumor agent”.

The term “aminopyridine derivative” as used in the specificationincludes, but is not limited to, any compound having a pyridyl group ora pyridine analogue group, any of which is substituted with an aminogroup. It is exemplified by a compound of the above Formula (I), andpreferably any one compound of the below-mentioned (a) to (c): acompound which is:

(a)(2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate,

(b) (2S)-1,1,1-trifluoropropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate,or

(c) 1,1,1-trifluoro-2-methylpropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate,or a pharmaceutically acceptable salt or ester thereof.

Embodiments of the compound represented by the above Formula (I) will beillustrated in more detail.

n is 0 or 1;

X is 0 or CH₂;

R¹ is H or C₁₋₂ alkyl.

Preferably, R¹ is H or methyl.

R² is H or C₁₋₃ alkyl.

Preferably, R² is H or methyl.

R³ and R⁴ are each independently H or C₁₋₂ alkyl, where the alkyl may besubstituted with one to three of the same or different substituentsselected from R¹⁰.

Preferably, R³ and R⁴ are each independently H or methyl which may besubstituted with one to three of the same or different substituentsselected from R¹⁰.

R⁵ is H or OCH₃.

Preferably, R⁵ is H.

R¹° is F or Cl.

Preferably, R¹⁰ is F.

Further, in the combined preparation comprising two separatepreparations according to the invention, preferably either or both ofthe two separate preparations are an oral preparation.

The combined preparation comprising two separate preparations accordingto the invention is preferably such that one of the preparations is apreparation containing, together with a pharmaceutically acceptablecarrier or diluent, the following:

(a) (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate,

(b) (2S)-1,1,1-trifluoropropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate,or

(c) 1,1,1-trifluoro-2-methylpropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate,

or a pharmaceutically acceptable salt or ester thereof; and

the other preparation is a preparation containing paclitaxel ordocetaxel, or a pharmaceutically acceptable salt or ester thereof,together with a pharmaceutically acceptable carrier or diluent.

Moreover, the combined preparation comprising, together with apharmaceutically acceptable carrier or diluent, two separatepreparations according to the invention may be further combined with atleast one preparation containing an antitumor agent selected from thegroup consisting of antitumor alkylating agents, antitumorantimetabolites, antitumor antibiotics, plant-derived antitumor agents,antitumor platinum coordination compounds, antitumor camptothecinderivatives, antitumor tyrosine kinase inhibitors, monoclonalantibodies, interferons, biological response modifiers and otherantitumor agents (here, definition of each antitumor agent is the sameas that defined above), or a pharmaceutically acceptable salt or esterthereof.

Also, the pharmaceutical composition according to the inventionpreferably contains, together with a pharmaceutically acceptable carrieror diluent, the following:

(a) (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate,

(b) (2S)-1,1,1-trifluoropropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate,or

(c) 1,1,1-trifluoro-2-methylpropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate,

or a pharmaceutically acceptable salt or ester thereof; and paclitaxelor docetaxel, or a pharmaceutically acceptable salt or ester thereof,together with a pharmaceutically acceptable carrier or diluent.

Description of the Process for Preparation of Compound of Formula (I)

Compounds represented by the Formula (I):

(wherein R₁, R₂, R₃, R₄ and R₅ have the same meaning as the symbols forthe above Formula (I)) can be prepared by, for example, the followingmethod. Hereinafter, the phrase “symbols for the above Formula (I)” asused herein means “the respective symbols as described for Formula (I)initially described in the present specification.”

(Process 1)

The present process is a method of subjecting the Compound (II) (whereinR₃ and R₄ have the same meaning as the symbols for the above Formula(I)) and 4-nitrophenyl chloroformate, to an O-acylation reaction,thereby to produce Compound (III) (wherein R₃ and R₄ have the samemeaning as the symbols for the above Formula (I)).

The Compound (II) used in this process may be exemplified by2-trifluoromethyl-2-propanol, 1,1,1-trifluoro-2-propanol, and the like.The Compound (II) is commercially available or can be prepared by aknown method.

The O-acylation reaction used in this process employs methods well knownto those skilled in the art. In the O-acylation reaction used in thisprocess, specifically, for example, the Compound (III) can besynthesized by reacting the Compound (II) in a solvent such aschloroform with a base such as pyridine, followed by adding thereto4-nitrophenyl chloroformate. In this reaction, 4-nitrophenylchloroformate is used in an amount of from 1 to 10 mol, preferably from1 to 5 mol; and the base is used in an amount of from 1 to 10 mol,preferably from 1 to 5 mol, relative to 1 mol of Compound (II). Thereaction temperature can be appropriately selected by a person havingordinary skill in the art in accordance with the starting compound orreaction solvent used, but it is typically from room temperature to 60°C. Also, the reaction is typically completed within 1 hour to 48 hours,but the reaction time can be appropriately extended or reduced.

The resulting Compound (III) is subjected to isolation and purificationby known separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

(Process 2)

The present process is a method of subjecting the Compound (III)(wherein R₃ and R₄ have the same meaning as the symbols for the aboveFormula (I)), obtained in the above-described Process 1, and Compound(IV) (wherein PG is a protective group such as benzyl andtert-butoxycarbonyl, and R₅ have the same meaning as the symbols for theabove Formula (I)), to an alkoxycarbonylation reaction, thereby toproduce Compound (V) (wherein PG is a protective group such as benzyland tert-butoxycarbonyl, and R₃, R₄ and R₅ have the same meaning as thesymbols for the above Formula (I)).

The Compound (IV) used in this process may be exemplified by(3S)-(+)-1-benzyl-3-aminopyrrolidine, tert-butyl(3R,4R)-3-amino-4-hydroxypyrrolidine-1-carboxylate, and the like. TheCompound (IV) is commercially available or can be prepared by a knownmethod.

The alkoxycarbonylation reaction used in this process employs methodswell known to those skilled in the art. In the alkoxycarbonylationreaction used in this process, specifically, for example, the Compound(V) can be synthesized by reacting the Compound (III) in a solvent suchas chloroform with a base such as N,N-diisopropylethylamine, followed byadding thereto the Compound (IV). In this reaction, Compound (IV) isused in an amount of from 1 to 10 mol, preferably from 1 to 5 mol; andthe base is used in an amount of from 1 to 10 mol, preferably from 1 to5 mol, relative to 1 mol of Compound (III). The reaction temperature canbe appropriately selected by a person having ordinary skill in the artin accordance with the starting compound or reaction solvent used, butit is typically from room temperature to 60° C. Also, the reaction istypically completed within 1 hour to 48 hours, but the reaction time canbe appropriately extended or reduced.

The resulting Compound (V) is subjected to isolation and purification byknown separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

(Process 3)

The present process is a method of deprotecting a protective group PG ofthe Compound

(V) (wherein PG is a protective group such as benzyl andtert-butoxycarbonyl, and R₃, R₄ and R₅ have the same meaning as thesymbols for the above Formula (I)), obtained in the above-describedProcess 2, thereby to produce Compound (VI) (wherein R₃, R₄ and R₅ havethe same meaning as the symbols for the above Formula (I)).

For the deprotection reaction of PG, the method may vary depending onthe type of the protective group or stability of the compound, butmethods described in the literature [See T.W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons (1981)] or methods equivalentthereto can be carried out. For example, the Compound (V) in which PG istert-butoxycarbonyl can be deprotected in a mixed solvent oftrifluoroacetic acid and chloroform. The reaction temperature can beappropriately selected by a person having ordinary skill in the art inaccordance with the starting compound or reaction solvent used, but itis typically from 0° C. to the boiling point of the solvent. Also, thereaction is typically completed between 1 hour to 24 hours, but thereaction time can be appropriately extended or reduced.

The resulting Compound (VI) is subjected to isolation and purificationby known separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

(Process 4)

The present process is a method of subjecting the Compound (VI) (whereinR₃, R₄ and R₅ have the same meaning as the symbols for the above Formula(I)), obtained in the above-described Process 3, and Compound (VII)(wherein R₁ and R₂ have the same meaning as the symbols for the aboveFormula (I)), to an amination reaction, thereby to produce Compound (I)(wherein R₁, R₂, R₃, R₄ and R₅ have the same meaning as the symbols forthe above Formula (I)).

The Compound (VII) used in this process may be exemplified by,2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine,2-chloro-6-methyl-N-(1H-pyrazol-3-yl)pyrimidin-4-amine, and the like.The Compound (VII) can be prepared by a known method.

The amination reaction used in this process employs methods well knownto those skilled in the art. In the amination reaction used in thisprocess, specifically, for example, the Compound (I) can be synthesizedby reacting the Compound (VI) in a solvent such as dimethylsulfoxidewith a base such as N,N-diisopropylethylamine, followed by addingthereto the Compound (VII). In this reaction, Compound (VII) is used inan amount of from 1 to 10 mol, preferably from 1 to 5 mol; and the baseis used in an amount of from 1 to 10 mol, preferably from 1 to 5 mol,relative to 1 mol of Compound (VI). The reaction temperature can beappropriately selected by a person having ordinary skill in the art inaccordance with the starting compound or reaction solvent used, but itis typically from room temperature to 100° C. Also, the reaction istypically completed within 1 hour to 48 hours, but the reaction time canbe appropriately extended or reduced.

The resulting Compound (I) is subjected to isolation and purification byknown separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

Compounds represented by the Formula (I) (wherein R₁, R₂, R₃, R₄ and R₅have the same meaning as the symbols for the above Formula (I)) can bealso prepared by, for example, the following method.

(Process 5)

The present process is a method of subjecting the Compound (VII)(wherein R₁ and R₂ have the same meaning as the symbols for the aboveFormula (I)) and Compound (VIII) (wherein R₅ has the same meaning as thesymbols for the above Formula (I)), to an amination reaction, thereby toproduce Compound (IX) (wherein R₁, R₂ and R₅ have the same meaning asthe symbols for the above Formula (I)).

The Compound (VII) used in this process may be exemplified by,2-chloro-6-methyl-N-(5-methyl-1H-pyrazol-3-yl)pyrimidin-4-amine,2-chloro-6-methyl-N-(1H-pyrazol-3-yl)pyrimidin-4-amine, and the like.The Compound (VII) can be prepared by a known method.

The Compound (VIII) used in this process may be exemplified by,(3S,4R)-4-methylpyrrolidin-3-amine,(4aR,7aR)-octahydropyrrolo[3,4-b][1,4]oxazine, and the like. TheCompound (IV) is commercially available or can be prepared by a knownmethod.

The amination reaction used in this process employs methods well knownto those skilled in the art. In the amination reaction used in thisprocess, specifically, for example, the Compound (IX) can be synthesizedby reacting the Compound (VII) in a solvent such as dimethylsulfoxidewith a base such as N,N-diisopropylethylamine, followed by addingthereto the Compound (VIII). In this reaction, Compound (VIII) is usedin an amount of from 1 to 10 mol, preferably from 1 to 5 mol; and thebase is used in an amount of from 1 to 10 mol, preferably from 1 to 5mol, relative to 1 mol of Compound (IV). The reaction temperature can beappropriately selected by a person having ordinary skill in the art inaccordance with the starting compound or reaction solvent used, but itis typically from room temperature to 120° C. Also, the reaction istypically completed within 1 hour to 48 hours, but the reaction time canbe appropriately extended or reduced.

The resulting Compound (IX) is subjected to isolation and purificationby known separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

(Process 6)

The present process is a method of subjecting the Compound (IX) (whereinR₁, R₂ and R₅ have the same meaning as the symbols for the above Formula(I)), obtained in the above-described Process 5, and Compound (III)(wherein R₃ and R₄ have the same meaning as the symbols for the aboveFormula (I)), obtained in the above-described Process 1, to andi-alkoxycarbonylation reaction, thereby to produce Compound (X)(wherein R₁, R₂, R₃, R₄ and R₅ have the same meaning as the symbols forthe above Formula (I)).

The di-alkoxycarbonylation reaction used in this process employs methodswell known to those skilled in the art. In the di-alkoxycarbonylationreaction used in this process, specifically, for example, the Compound(X) can be synthesized by reacting the Compound (IX) in a solvent suchas chloroform with a base such as N,N-diisopropylethylamine, followed byadding thereto the Compound (III). In this reaction, Compound (III) isused in an amount of from 2 to 20 mol, preferably from 2 to 10 mol; andthe base is used in an amount of from 2 to 20 mol, preferably from 2 to10 mol, relative to 1 mol of Compound (IX). The reaction temperature canbe appropriately selected by a person having ordinary skill in the artin accordance with the starting compound or reaction solvent used, butit is typically from room temperature to 60° C. Also, the reaction istypically completed within 1 hour to 48 hours, but the reaction time canbe appropriately extended or reduced.

The resulting Compound (X) is subjected to isolation and purification byknown separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

(Process 7)

The present process is a method for the hydrolysis of the Compound (X)(wherein R₁, R₂, R₃, R₄ and R₅ have the same meaning as the symbols forthe above Formula (I)), obtained in the above-described Process 6,thereby to produce Compound (I) (wherein R₁, R₂, R₃, R₄ and R₅ have thesame meaning as the symbols for the above Formula (I)).

The hydrolysis used in this process employs methods well known to thoseskilled in the art. In the hydrolysis reaction used in this process,specifically, for example, the Compound (I) can be synthesized byreacting the Compound (X) in a solvent such as methanol with a base suchas potassium carbonate. In this reaction, the base is used in an amountof from 2 to 20 mol, preferably from 2 to 10 mol, relative to 1 mol ofCompound (I). The reaction temperature can be appropriately selected bya person having ordinary skill in the art in accordance with thestarting compound or reaction solvent used, but it is typically from 0°C. to room temperature. Also, the reaction is typically completed within1 hour to 48 hours, but the reaction time can be appropriately extendedor reduced.

The resulting Compound (I) is subjected to isolation and purification byknown separation and purification means such as, for example,concentration, concentration under reduced pressure, crystallization,solvent extraction, reprecipitation or chromatography, or may besubjected to the next process without isolation and purification.

Next, the Aurora A and Aurora B inhibitory actions of the compound ofGeneral Formula (I) according to the invention will be explained below.

Aurora A Inhibitory Action

(1) Purification of Aurora A

cDNA of Aurora A having histidine tag fused at the amino terminal wasintegrated into an expression vector, which was then highly expressed inEscherichia coli BL21-CodonPlus(DE3)-RIL cells. The Escherichia colicells were recovered and solubilized, and then the histidine-taggedAurora A protein was adsorbed onto a nickel chelate column and elutedfrom the column with imidazole. The active fraction was desalted with adesalting column to give a pure enzyme.

(2) Measurement of activity of Aurora A

For measurement of the activity of Aurora A, the substrate used wasKemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (SEQ. ID. NO.: 1), a syntheticpeptide purchased from Sigma-Aldrich, Inc. [Certificate of analysis(Upstate, Inc.)].

Reaction was conducted by a partial modification of a method by Upstate,Inc. [Kinase Profiler™ Assay Protocols]. The amount of the reactionliquid was 21.1 μL, and the composition of the reaction buffer (R2buffer) was 50 mM Tris-hydrochloride buffer (pH 7.4)/15 mM magnesiumacetate/0.2 mM ethylenediamine-N,N,N′,N′-tetraacetate (EDTA). To this,purified Aurora A, 100 μM of a substrate peptide, 20 μM of unlabeledadenosine triphosphate (ATP) and 0.5 μCi of [γ-³³P] labeled ATP (2,500Ci/mmole or more) were added, and the mixture was reacted at 30° C. for20 minutes. Then, 10 μL of 350 mM phosphate buffer was added to thereaction system to stop the reaction. The substrate peptide was adsorbedon a P81 paper filter 96-well plate and then washed with 130 mMphosphate buffer for several times. The radiation activity of thepeptide was measured with a liquid scintillation counter. The [γ-³³P]labeled ATP was purchased from Amersham Biosciences Co., Ltd.

The compound to be tested was added to the reaction system such that adilution series of the compound in dimethylsulfoxide was first prepared,and 1.1 μL of this solution was added. A control was provided by adding1.1 μL of DMSO to the reaction system.

Aurora B Inhibitory Action

(1) Purification of Aurora B

cDNA of Aurora B having histidine tag fused at the amino terminal wasintegrated into an expression vector, which was then highly expressed inEscherichia coli BL21-CodonPlus(DE3)-RIL cells. The Escherichia colicells were recovered and solubilized, and then the histidine-taggedAurora A protein was adsorbed onto a nickel chelate column and elutedfrom the column with imidazole. The active fraction was desalted with adesalting column to give a pure enzyme.

(2) Measurement of activity of Aurora B

For measurement of the activity of Aurora B, the substrate used wasKemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) (SEQ. ID. NO.: 1), a syntheticpeptide purchased from Sigma-Aldrich, Inc. [Certificate of analysis(Upstate, Inc.)].

Reaction was conducted by a partial modification of the method ofactivity measurement for Aurora A. The amount of the reaction liquid was21.1 μL, and the composition of the reaction buffer (R2 buffer) was 50mM Tris-hydrochloride buffer (pH 7.4)/15 mM magnesium acetate/0.2 mMethylenediamine-N,N,N,N-tetraacetate (EDTA). To this, purified Aurora B,100 μM of a substrate peptide, 100 μM of unlabeled adenosinetriphosphate (ATP) and 1 μCi of [γ-³³P] labeled ATP (2,500 Ci/mmole ormore) were added, and the mixture was reacted at 30° C. for 20 minutes.Then, 10 μL of 350 mM phosphate buffer was added to the reaction systemto stop the reaction. The substrate peptide was adsorbed on a P81 paperfilter 96-well plate and then washed with 130 mM phosphate buffer forseveral times. The radiation activity of the peptide was measured with aliquid scintillation counter. The [γ-³³P] labeled ATP was purchased fromAmersham Biosciences Co., Ltd.

TABLE 1 Inhibitory activity Inhibitory activity for Aurora A for AuroraB Example (IC₅₀, nM) (IC₅₀, nM) Example 1 2.3 730 Example 2 1.3 85Example 3 5.4 870

From the above, the compound according to the invention is believed tobe useful as an antitumor agent since it exhibits excellent Aurora Aselective inhibitory activity, leading to a synergistic action incombined use with other antitumor agent. Thus, it is believed that apharmaceutical composition or Aurora A selective inhibitor containingthe novel aminopyridine derivative according to the invention or apharmaceutically acceptable salt or ester thereof, or an antitumor agentcontaining the compound according to the invention or a pharmaceuticallyacceptable salt or ester thereof is effective in the treatment of cancerpatients.

The above-mentioned pharmaceutical composition and inhibitor, and theabove-mentioned antitumor agent may contain a pharmaceuticallyacceptable carrier or diluent. Here, the “pharmaceutically acceptablecarrier or diluent” refers to excipients [e.g., fats, beeswax,semi-solid and liquid polyols, natural or hydrogenated oils, etc.];water (e.g., distilled water, particularly distilled water forinjection, etc.), physiological saline, alcohol (e.g., ethanol),glycerol, polyols, aqueous glucose solution, mannitol, plant oils,etc.); additives [e.g., extending agent, disintegrating agent, binder,lubricant, wetting agent, stabilizer, emulsifier, dispersant,preservative, sweetener, colorant, seasoning agent or aromatizer,concentrating agent, diluent, buffer substance, solvent or solubilizingagent, chemical for achieving storage effect, salt for modifying osmoticpressure, coating agent or antioxidant], and the like.

A suitable tumor for which the therapeutic effect of the compoundaccording to the invention is expected may be exemplified by human solidcancer. Examples of human solid cancer include brain cancer, head andneck cancer, esophageal cancer, thyroid cancer, small cell carcinoma,non-small cell carcinoma, breast cancer, stomach cancer, gallbladder andbile duct cancer, liver cancer, pancreas cancer, colon cancer, rectalcancer, ovarian cancer, chorioepithelioma, uterine cancer, cervicalcancer, renal pelvic and ureteral cancer, bladder cancer, prostatecancer, penile cancer, testicular cancer, embryonal cancer, Wilms'tumor, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma,Ewing's tumor, soft tissue sarcoma, and the like.

Next, the above-described “pharmaceutically acceptable salt or ester”will be explained below.

When the compound according to the invention is used as an antitumoragent or the like, it may be also used in a form of pharmaceuticallyacceptable salt. Typical examples of the pharmaceutically acceptablesalt include a salt with an alkali metal such as sodium and potassium; asalt with an inorganic acid, such as hydrochloride, sulfate, nitrate,phosphate, carbonate, hydrogen carbonate, and perchlorate; a salt withan organic acid, such as acetate, propionate, lactate, maleate,fumarate, tartrate, malate, citrate, and ascorbate; a salt with sulfonicacid, such as methanesulfonate, isethionate, benzenesulfonate, andtoluenesulfonate; a salt with acidic amino acid, such as aspartate andglutamate; and the like. A pharmaceutically acceptable salt of theCompound (I) is preferably a salt with an inorganic acid, such ashydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogencarbonate, and perchlorate; more preferably hydrochloride.

The process for preparation of a pharmaceutically acceptable salt of thecompound according to the invention may be carried out by an appropriatecombination of those methods that are conventionally used in the fieldof organic synthetic chemistry. A specific example thereof is a methodin which a solution of the compound according to the invention in itsfree form is subjected to neutralization titration with an alkalinesolution or an acidic solution.

Examples of the ester of the compound according to the invention includemethyl ester and ethyl ester. Such esters can be prepared byesterification of a free carboxyl group according to a conventionalmethod.

With regard to each preparation of the combined preparation according tothe invention, various preparation forms can be selected, and examplesthereof include oral preparations such as tablets, capsules, powders,granules or liquids, or sterilized liquid parenteral preparations suchas solutions or suspensions, suppositories, ointments and the like.

Solid preparations can be prepared in the forms of tablet, capsule,granule and powder without any additives, or prepared using appropriatecarriers (additives). Examples of such carriers (additives) may includesaccharides such as lactose or glucose; starch of corn, wheat or rice;fatty acids such as stearic acid; inorganic salts such as magnesiummetasilicate aluminate or anhydrous calcium phosphate; syntheticpolymers such as polyvinylpyrrolidone or polyalkylene glycol; alcoholssuch as stearyl alcohol or benzyl alcohol; synthetic cellulosederivatives such as methylcellulose, carboxymethylcellulose,ethylcellulose or hydroxypropylmethylcellulose; and other conventionallyused additives such as gelatin, talc, plant oil and gum arabic.

These solid preparations such as tablets, capsules, granules and powdersmay generally contain, for example, 0.1 to 100% by weight, andpreferably 5 to 98% by weight, of the compound of the above Formula (I)as an active ingredient, based on the total weight of the preparation.

Liquid preparations are produced in the forms of suspension, syrup,injection and drip infusion (intravenous fluid) using appropriateadditives that are conventionally used in liquid preparations, such aswater, alcohol or a plant-derived oil such as soybean oil, peanut oiland sesame oil.

In particular, when the preparation is administered parenterally in aform of intramuscular injection, intravenous injection or subcutaneousinjection, appropriate solvent or diluent may be exemplified bydistilled water for injection, an aqueous solution of lidocainehydrochloride (for intramuscular injection), physiological saline,aqueous glucose solution, ethanol, polyethylene glycol, propyleneglycol, liquid for intravenous injection (e.g., an aqueous solution ofcitric acid, sodium citrate and the like) or an electrolytic solution(for intravenous drip infusion and intravenous injection), or a mixedsolution thereof.

Such injection may be in a form of a preliminarily dissolved solution,or in a form of powder per se or powder associated with a suitablecarrier (additive) which is dissolved at the time of use. The injectionliquid may contain, for example, 0.1 to 10% by weight of an activeingredient based on the total weight of the preparation.

Liquid preparations such as suspension or syrup for oral administrationmay contain, for example, 0.1 to 10% by weight of an active ingredientbased on the total weight of the preparation.

Each preparation of the combined preparation according to the inventioncan be prepared by a person having ordinary skill in the art accordingto conventional methods or common techniques. For example, a preparationcontaining another antitumor agent that is used in combination with thecompound represented by the above Formula (I), can be prepared, if thepreparation is an oral preparation, for example, by mixing anappropriate amount of the antitumor agent with an appropriate amount oflactose and filling this mixture into hard gelatin capsules which aresuitable for oral administration. On the other hand, preparation can becarried out, if the preparation containing the antitumor agent is aninjection, for example, by mixing an appropriate amount of the antitumoragent with an appropriate amount of 0.9% physiological saline andfilling this mixture in vials for injection.

Also, in the case of a combination preparation containing the compoundrepresented by the above Formula (I) according to the invention andanother antitumor agent, a person having ordinary skill in the art caneasily prepare the preparation according to conventional methods orcommon techniques.

In the process according to the invention, preferred therapeutic unitmay vary in accordance with, for example, the administration route ofthe compound represented by the Formula (I), the type of the compoundrepresented by the Formula (I) used, and the dosage form of the compoundrepresented by the Formula (I) used; the type, administration route anddosage form of the other antitumor agent used in combination; and thetype of cells to be treated, the condition of patient, and the like. Theoptimal treatment under the given conditions can be determined by aperson skilled in the art, based on the set conventional therapeuticunit and/or based on the content of the present specification.

In the process according to the invention, the therapeutic unit for thecompound represented by the above Formula (I) may vary in accordancewith, specifically, the type of compound used, the type of compoundedcomposition, application frequency and the specific site to be treated,seriousness of the disease, age of the patient, doctor's diagnosis, thetype of cancer, or the like. However, as an exemplary reference, thedaily dose for an adult may be within a range of, for example, 1 to1,000 mg in the case of oral administration. In the case of parenteraladministration, preferably intravenous administration, and morepreferably intravenous drip infusion, the daily dose may be within arange of, for example, 1 to 100 mg/m² (body surface area). Here, in thecase of intravenous drip infusion, administration may be continuouslycarried out for, for example, 1 to 48 hours. Moreover, theadministration frequency may vary depending on the administering methodand symptoms, but it is, for example, once to five times a day.Alternatively, periodically intermittent administration such asadministration every other day, administration every two days or thelike may be employed as well in the administering method. The period ofwithdraw from medication in the case of parenteral administration is,for example, 1 to 6 weeks.

Although the therapeutic unit for the other antitumor agent used incombination with the compound represented by the Formula (I) is notparticularly limited, it can be determined, if needed, by those skilledin the art according to known literatures. Examples may be as follows.

The therapeutic unit of 5-fluorouracil (5-FU) is such that, in the caseof oral administration, for example, 200 to 300 mg per day isadministered in once to three times consecutively, and in the case ofinjection, for example, 5 to 15 mg/kg per day is administered once a dayfor the first 5 consecutive days by intravenous injection or intravenousdrip infusion, and then 5 to 7.5 mg/kg is administered once a day everyother day by intravenous injection or intravenous drip infusion (thedose may be appropriately increased or decreased).

The therapeutic unit of S-1 (Tegafur, Gimestat and Ostat potassium) issuch that, for example, the initial dose (singe dose) is set to thefollowing standard amount in accordance with the body surface area, andit is orally administered twice a day, after breakfast and after dinner,for 28 consecutive days, followed by withdrawal from medication for 14days. This is set as one course of administration, which is repeated.The initial standard amount per unit body surface area (Tegafurequivalent) is 40 mg in one administration for an area less than 1.25m²; 50 mg in one administration for an area of 1.25 m² to less than 1.5m²; 60 mg in one administration for an area of 1.5 m² or more. This doseis appropriately increased or decreased depending on the condition ofthe patient.

The therapeutic unit for gemcitabine is, for example, 1 g asgemcitabine/m² in one administration, which is administered byintravenous drip infusion over a period of 30 minutes, and oneadministration per week is continued for 3 weeks, followed by withdrawalfrom medication on the fourth week. This is set as one course ofadministration, which is repeated. The dose is appropriately decreasedin accordance with age, symptom or development of side-effects.

The therapeutic unit for doxorubicin (e.g., doxorubicin hydrochloride)is such that, for example, in the case of intravenous injection, 10 mg(0.2 mg/kg) (titer) is administered once a day by intravenous one-shotadministration for 4 to 6 consecutive days, followed by withdrawal frommedication for 7 to 10 days. This is set as one course ofadministration, which is repeated two or three times. Here, the totaldose is preferably 500 mg (titer)/m² (body surface area) or less, and itmay be appropriately increased or decreased within the range.

The therapeutic unit for etoposide is such that, for example, in thecase of intravenous injection, 60 to 100 mg/m² (body surface area) perday is administered for 5 consecutive days, followed by withdrawal frommedication for three weeks (the dose may be appropriately increased ordecreased). This is set as one course of administration, which isrepeated. Meanwhile, in the case of oral administration, for example,175 to 200 mg per day is administered for 5 consecutive days, followedby withdrawal from medication for three weeks (the dose may beappropriately increased or decreased). This is set as one course ofadministration, which is repeated.

The therapeutic unit for docetaxel (docetaxel hydrate) is such that, forexample, 60 mg as docetaxel/m² (body surface area) is administered oncea day by intravenous drip infusion over a period of 1 hour or longer atan interval of 3 to 4 weeks (the dose may be appropriately increased ordecreased).

The therapeutic unit of paclitaxel is such that, for example, 210 mg/m²(body surface area) is administered once a day by intravenous dripinfusion over a period of 3 hours, followed by withdrawal frommedication for at least 3 weeks. This is set as one course ofadministration, which is repeated. The dose may be appropriatelyincreased or decreased.

The therapeutic unit for cisplatin is such that, for example, in thecase of intravenous injection, 50 to 70 mg/m² (body surface area) isadministered once a day, followed by withdrawal from medication for 3weeks or longer (the dose may be appropriately increased or decreased).This is set as one course of administration, which is repeated.

The therapeutic unit for carboplatin is such that, for example, 300 to400 mg/m² is administered once a day by intravenous drip infusion over aperiod of 30 minutes or longer, followed by withdrawal from medicationfor at least 4 weeks (the dose may be appropriately increased ordecreased). This is set as one course of administration, which isrepeated.

The therapeutic unit for oxaliplatin is such that 85 mg/m² isadministered once a day by intravenous injection, followed by withdrawalfrom medication for two weeks. This is set as one course ofadministration, which is repeated.

The therapeutic unit for irinotecan (e.g., irinotecan hydrochloride) issuch that, for example, 100 mg/m² is administered once a day byintravenous drip infusion for 3 or 4 times at an interval of one week,followed by withdrawal from medication for at least two weeks.

The therapeutic unit for topotecan is such that, for example, 1.5 mg/m²is administered once a day by intravenous drip infusion for 5 days,followed by withdrawal from medication for at least 3 weeks.

The therapeutic unit for cyclophosphamide is such that, for example, inthe case of intravenous injection, 100 mg is administered once a day byintravenous injection for consecutive days. If the patient can tolerate,the daily dose may be increased to 200 mg. The total dose is 3,000 to8,000 mg, which may be appropriately increased or decreased. Ifnecessary, it may be injected or infused intramuscularly,intrathoracically or intratumorally. On the other hand, in the case oforal administration, for example, 100 to 200 mg is administered a day.

The therapeutic unit for gefitinib is such that 250 mg is orallyadministered once a day.

The therapeutic unit for cetuximab is such that, for example, 400 mg/m²is administered on the first day by intravenous drip infusion, and then250 mg/m² is administered every week by intravenous drip infusion.

The therapeutic unit for bevacizumab is such that, for example, 3 mg/kgis administered every week by intravenous drip infusion.

The therapeutic unit for trastuzumab is such that, for example,typically for an adult, once a day, 4 mg as trastuzumab/kg (body weight)is administered initially, followed by intravenous drip infusion of 2mg/kg over a period of 90 minutes or longer every week from the secondadministration.

The therapeutic unit for exemestane is such that, for example, typicallyfor an adult, 25 mg is orally administered once a day after meal.

The therapeutic unit for leuprorelin (e.g., leuprorelin acetate) is suchthat, for example, typically for an adult, 11.25 mg is subcutaneouslyadministered once in 12 weeks.

The therapeutic unit for imatinib is such that, for example, typicallyfor an adult in the chronic phase of chronic myelogenous leukemia, 400mg is orally administered once a day after meal.

The therapeutic unit for a combination of 5-FU and leucovorin is suchthat, for example, 425 mg/m² of 5-FU and 200 mg/m² of leucovorin areadministered from the first day to the fifth day by intravenous dripinfusion, and this course is repeated at an interval of 4 weeks.

The therapeutic unit for sorafenib is such that, for example, 200 mg isorally administered twice a day (400 mg per day) at least 1 hour beforeor 2 hours after eating.

The therapeutic unit for sunitinib is such that, for example, 50 mg isorally administered once a day for four weeks, followed by 2 weeks off.

WORKING EXAMPLES

In a thin-layer chromatography of Examples and Referential Examples,Silica gel 60 F254 (Merck) and Chromatolex NH (Fuji Silysia Chemical)was used as a plate and a UV detector was used in a detecting method. Aspre-packed silica gel column for the chromatography, Biotage KP-SilFLASH Cartridge (Biotage) or Purif-Pack Si (Moritex) were used. AndKP-NH FLASH Cartridge (Biotage) or Purif-Pack NH (Moritex) was used forbasic silica gel column chromatography. In a reversed phase preparativehigh performance liquid chromatography, XBridge Prep C18 (30×50 mm)(Waters) was used as a column, and a 0.1% aqueous trifluoroacetic acidsolution and a 0.1% solution of trifluoroacetic acid in acetonitrilewere used in a mobile phase. ESI-MS spectra were measured usingmicromass ZQ (Micromass). NMR spectra were measured using a spectrometerin a type of AL 400 (400 MHz; JEOL) and Inova 600 (600 MHz; Varian). Formicrowave reaction was used Initiator (Biotage).

Meanings of abbreviations used in the NMR measurement are as follows.

-   s: singlet-   d: doublet-   dd: double doublet-   t: triplet-   dt: double triplet-   tt: triple triplet-   q: quartet-   m: multiplet-   br: broad-   brs: broad singlet-   Hz: hertz-   DMSO-d₆: dimethylsulfoxide-d₆-   CDCl₃: chloroform-d-   CD₃OD: methanol-d₄    Meanings of abbreviations used in experimental section are as    follows.-   Boc: tert-butoxycarbonyl group-   dba: dibenzylideneacetone-   DIEA: N,N-diisopropylethylamine-   DMF: N,N-dimethylformamide-   DMSO: dimethylsulfoxide-   dppf: 1,1′-bis(diphenylphosphino)ferrocene-   EDCI.HCl: N-[2-(dimethylamino)ethyl]-M-ethylcarbodiimide    hydrochloride-   EtOAc: ethyl acetate-   HOBt.H₂O: 1H-benzotriazole monohydrate-   Me: methyl group-   MeOH: methanol-   Ms: methanesulfonyl group-   MSCl: methanesulfonyl chloride-   NMP: N-methylpyrolidone-   PPh₃: triphenylphosphine-   RP-HPLC: reverse phase high performance liquid chromatography-   PTLC: preparative thin layer chromatography-   TEA: triethylamine-   TPA: 2,2,2-trifluoroacetic acid-   THF: tetrahydrofuran

Example 1 Preparation of (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-metboxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate

Step 1: Preparation of tert-butyl(3S)-3-(tert-butyldimethylsilyl)oxy-4-oxopyrrolidine-1-carboxylate

To a solution of oxalyl chloride (0.51 mL) in CH₂Cl₂ (7.3 mL) was addeda 4.0M CH₂Cl₂ solution of DMSO (0.86 mL) at −78° C. After 10 min, asolution of tert-butyl(3S,4S)-3-hydroxy-4-(tert-butyldimethylsilyl)oxypyrrolidine-1-carboxylate(920 mg) in CH₂Cl₂ (5.8 mL) was added, and the mixture was stirred for 2hours at −45° C. Triethylamine (3.25 mL) was added, and after beingstirred for 10 min at room temperature, water was added. The mixture wasseparated, and the aqueous layer was extracted with ether. The combinedorganic layer was washed with brine, and dried over MgSO₄ andconcentrated. The residue was purified with silica gel columnchromatography (eluent: hexane/EtOAc=1/0˜50/50) to give tert-butyl(3S)-3-(tert-butyldimethylsilyl)oxy-4-oxopyrrolidine-1-carboxylate as apale yellow foam.

Step 2: Preparation of tert-butyl(3R,4S)-3-hydroxy-4-(tert-butyldimethylsilyl)oxy-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylate

To an ice-cooled solution of tert-butyl(3S)-3-(tert-butyldimethylsilyl)oxy-4-oxopyrrolidine-1-carboxylate (1.2g) in THF (5 mL) was added dropwise a 1M solution of allylmagnesiumbromide in THF (3.8 mL), and the mixture was stirred for 30 min at 0° C.The reaction was quenched with saturated aqueous NH₄Cl (5 mL) and themixture was extracted with Et₂O. The extract was washed with brine,dried over MgSO₄, and concentrated in vacuo. The residue was purifiedwith silica gel column chromatography (eluent: hexane/EtOAc=1/0˜50/50)to give tert-butyl(3R,4S)-3-hydroxy-4-(tert-butyldimethylsilyl)oxy-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylateas a pale yellow foam.

Step 3: Preparation of tert-butyl(3R,4S)-4-(tert-butyldimethylsilyl)oxy-3-methoxy-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylate

To a solution of tert-butyl(3R,4S)-3-hydroxy-4-(tert-butyldimethylsilyl)oxy-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylate(550 mg) in DMF (10 mL) was added iodemethane (2.2 g) at 0° C. After 10min, 60% NaH in oil (62 mg) was added, and the mixture was stirred for 1hour at room temperature. The reaction was quenched with saturatedaqueous NH₄Cl and the mixture was extracted with Et₂O. The extract waswashed with brine, dried over MgSO₄, and concentrated in vacuo. Theresidue was purified with silica gel column chromatography (eluent:hexane/EtOAc=1/0˜50/50) to give tert-butyl(3R,4S)-4-(tert-butyldimethylsilyl)oxy-3-methoxy-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylateas a pale yellow foam.

Step 4: Preparation of tert-butyl(3R,4S)-4-(tert-butyldimethylsilyl)oxy-3-(2-hydroxyethyl)-3-methoxypyrrolidine-1-carboxylate

A solution of tert-butyl(3R,4S)-4-(tert-butyldimethylsilyl)oxy-3-methoxy-3-(prop-2-en-1-yl)pyrrolidine-1-carboxylate(230 mg) in CH₂Cl₂ (10 mL) was cooled to −78° C. and treated with astream of ozone until a blue color persisted. A stream of nitrogen waspassed through the solution for 15 minutes to remove excess ozone fromsolution. The solution was then treated with dimethylsulfide (0.2 mL)and allowed to warm to 23° C. over 30 min. Volatile components of thereaction mixture were removed in vacuo. The residue was dissolved inmethanol (10 mL). To the solution was added NaBH₄ (50 mg) at 0° C. andthe mixture was stirred at 0° C. for 30 minutes. The mixture was pouredinto saturated aqueous solution of NH₄Cl and extracted with CHCl₃. Theextract was washed with brine, dried over MgSO₄ and concentrated. Theresidue was purified with silica gel column chromatography (eluent:hexane/EtOAc=99/1˜1/99) to give tert-butyl(3R,4S)-4-(tert-butyldimethylsilyl)oxy-3-(2-hydroxyethyl)-3-methoxypyrrolidine-1-carboxylateas a pale yellow foam.

Step 5: Preparation of tert-butyl(3R,4S)-3-(2-azidoethyl)-4-hydroxy-3-methoxypyrrolidine-1-carboxylate

To the solution of tert-butyl(3R,4S)-4-(tert-butyldimethylsilyl)oxy-3-(2-hydroxyethyl)-3-methoxypyrrolidine-1-carboxylate(96 mg) in Et₂O (5 mL) was added DIEA (140 mg) and MSCl (60 mg) at roomtemperature. And the mixture was stirred at room temperature for 1 hour.After the period, the mixture was poured into saturated aqueous solutionof NaHCO₃ and extracted with CHCl₃. The extract was dried over Na₂SO₄and concentrated. The residue was dissolved into DMF (5 mL). To thesolution was added NaN₃ (50 mg) and the mixture was heated to 90° C.under stirring overnight. The mixture was poured into saturated aqueoussolution of NaHCO₃ and extracted with CHCl₃. The extract was dried overNa₂SO₄ and concentrated. The residue was dissolved in THF (2 mL), andthen the solution was added into tetrabutylammonium fluoride (1M THFsolution, 0.5 mL) under ice cooling. The mixture was stirred at roomtemperature at 2 hours, and then diluted with EtOAc and washed withbrine, dried over MgSO₄, and then concentrated in vacuo. The residue waspurified with silica gel column chromatography (eluent:hexane/EtOAc=99/1˜50/50) to give 1-tent-butyl 3-methyltrans-5-azidopiperidine-1,3-dicarboxylate as a pale yellow foam.

Step 6: Preparation of tert-butyl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate

To the solution of tert-butyl(3R,4S)-3-(2-azidoethyl)-4-hydroxy-3-methoxypyrrolidine-1-carboxylat (85mg) in CHCl₃ (5 mL) was added triethylamine (140 mg) and MsCl (50 mg) atroom temperature. And the mixture was stirred at room temperature for 1hour. After the period, the mixture was poured into saturated aqueoussolution of NaHCO₃ and extracted with CHCl₃. The extract was dried overNa₂SO₄ and concentrated. The residue was dissolved in THF (5 mL). To thesolution was added 10% palladium on activated carbon (20 mg). Theresulting solution was placed under a H₂ atmosphere (balloon pressure)and stirred at room temperature for 15 hours. The solution obtained byfiltration through Celite and washing the filter cake with MeOH wasconcentrated in vacuo. The residue was dissolved in CHCl₃ (3 mL) andrefluxed for 1 day and then cooled to room temperature. The resultingmixture was purified with silica gel column chromatography (eluent:CHCl₃/MeOH=99/1˜80/20) to give tert-butyl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate asa pale yellow foam.

Step 7: Preparation of 5-tert-butyl 1-[(2S)-1,1,1-trifluoropropan-2-yl](3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1,5-dicarboxylate

To a solution of 4-nitrophenyl (2S)-1,1,1-trifluoropropan-2-yl carbonate(15 mg) prepared in Referential Example 2 in CHCl₃ (2 mL) was addedtert-butyl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-5(1H)-carboxylate (11mg) at room temperature. The resulting mixture was stirred at 100° C. ina sealed tube for 2 hours and then cooled to room temperature. Themixture was purified with silica gel column chromatography (eluent:hexane/EtOAc=1/0˜0/1) to give 5-tert-butyl1-[(2S)-1,1,1-trifluoropropan-2-yl](3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1,5-dicarboxylate asa pale yellow foam.

Step 8: Preparation of (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate

To the solution of 5-tert-butyl 1-[(2S)-1,1,1-trifluoropropan-2-yl](3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1,5-dicarboxylate (15mg) in CHCl₃ (0.5 mL) was added TFA (0.5 mL). After stirring for 1 hourat room temperature, the mixture was poured into saturated aqueoussolution of NaHCO₃ and extracted with CHCl₃. The extract was dried overNa₂SO₄ and concentrated to give (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate asa pale yellow foam.

Step 9: Preparation of (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate

To a solution of (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate (8mg) in DMSO (1 mL) was added2-chloro-6-methyl-N-(1H-pyrazol-3-yl)pyrimidin-4-amine (6 mg) preparedin Referential Example 1 and DIEA (10 mg) at room temperature. Theresulting mixture was stirred at 120° C. for 15 hours and then cooled toroom temperature. The mixture was purified with preparative RP-HPLC togive (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b)]pyrrole-1(2H)-carboxylateas a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 1.30-1.45 (3H, m),1.95-2.31 (5H, m), 3.26 (3H, s), 3.40-4.20 (7H, m), 5.22-5.40 (1H, m),5.95-6.72 (2H, m), 7.40-7.70 (111, m), 9.35-9.58 (1H, m), 12.15 (1H,brs) ESI-MS m/z 456 [M+H]⁺.

Example 2 Preparation of (2S)-1,1,1-trifluoropropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate

Step 1: Preparation of2-[(4aR,7aR)-hexahydropyrrolo[3,4-b][1,4]oxazin-6(2H)-yl]-6-methyl-N-(1H-pyrazol-3-yl)pyrimidin-4-amine

The title compound was prepared by the similar manner described in Step9 of Example 1 by using (4aR,7aR)-octahydropyrrolo[3,4-b][1,4]oxazineinstead of (2S)-1,1,1-trifluoropropan-2-yl(3aR,6aR)-3a-methoxyhexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate.

Step 2: Preparation of (2S)-1,1,1-trifluoropropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate

To a mixture of2-[(4aR,7aR)-hexahydropyrrolo[3,4-b][1,4]oxazin-6(2H)-yl]-6-methyl-N-(1H-pyrazol-3-yl)pyrimidin-4-amine(20 mg) in CHCl₃ (3 mL) were added 4-nitrophenyl(2S)-1,1,1-trifluoropropan-2-yl carbonate (37 mg) prepared inReferential Example 2 and DIEA (30 mg) at room temperature. After 4-hourstirring at 100° C. in a sealed tube, the resulting mixture was cooledto room temperature and concentrated. The residue was diluted with MeOH(5 mL). To the resulting mixture was added potassium carbonate (100 mg)at 0° C. and stirred at 0° C. for 30 min. The mixture was poured intosaturated aqueous solution of NH₄Cl and extracted with CHCl₃. Theextract was washed with brine, dried over MgSO₄ and concentrated. Theresidue was purified with silica gel column chromatography (eluent:CHCl3/MeOH=99/1˜80/20) to give (2S)-1,1,1-trifluoropropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylateas a pale yellow foam. ESI-MS m/z 442 [M+H]⁺.

Example 3

1,1,1-trifluoro-2-methylpropan-2-yl(4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate

The title compound was prepared by the similar manner described in Step2 of Example 2 by using 4-nitrophenyl1,1,1-trifluoro-2-methylpropan-2-yl carbonate prepared in ReferentialExample 3 instead of 4-nitrophenyl (2S)-1,1,1-trifluoropropan-2-ylcarbonate: ESI-MS m/z 456 [M+H]⁺.

REFERENTIAL EXAMPLES Referential Example 1 Preparation of2-chloro-6-methyl-N-(1H-pyrazol-3-yl)pyrimidin-4-amine

To the solution of 2,4-dichloro-6-methylpyrimidine (3.00 g) and1H-pyrazol-3-amine (1.31 g) in NMP (30 mL) was added NaI (2.36 g) andDIEA (6.33 mL). And the mixture was heated to 80° C. under stirring.After 1 day, the mixture was cooled to room temperature. The mixture wasdiluted with Et₂O. The precipitate was collected by filtration, washedwith the mixed solvent of hexane and EtOAc and dried in vacuo to give2-chloro-6-methyl-N-(1H-pyrazol-5-yl)pyrimidin-4-amine (2.60 g) as apale yellow solid.

Referential Example 2 4-nitrophenyl (2S)-1,1,1-trifluoropropan-2-ylcarbonate

To a solution of (S)-(−)-1,1,1-trifluoro-2-propanol (1.95 g) in CHCl₃(30 mL) was added 4-nitrophenyl chloroformate (5.16 g) and pyridine(1.35 g) at room temperature. After 1-hour stirring at 60° C., theresulting mixture was cooled to room temperature. To the resultingmixture was added 2-(dimethylamino)ethanol (1.52 g) and was stirred atroom temperature for 1 hour. The mixture was poured into 1M aqueoussolution of HCl and extracted with CHCl₃. The extract was washed withwater and then dried over Na₂SO₄ and concentrated. The residue waspurified with silica gel column chromatography (eluent:hexane/EtOAc=1/0˜50/50) to give 4-nitrophenyl(2S)-1,1,1-trifluoropropan-2-yl carbonate as a pale yellow foam.

Referential Example 3 4-nitrophenyl 1,1,1-trifluoro-2-methylpropan-2-ylcarbonate

To a solution of 1,1,1-trifluoro-2-methylpropan-2-ol (1.0 g) in THF (30mL) was added a 2M solution of n-butyl lithium in n-hexane (3.9 mL) andthen 4-nitrophenyl chloroformate (2.4 g) at −75° C. The mixture waswarmed to room temperature and diluted with ethyl acetate, washed withwater, dried over sodium sulfate, filtered and concentrated in vacuo.The residue was purified by silica gel chromatography (0-50%EtOAc/Hexane) to give 4-nitrophenyl 1,1,1-trifluoro-2-methylpropan-2-ylcarbonate as a pale yellow foam.

INDUSTRIAL APPLICABILITY

The compound of the invention exhibits excellent Aurora A selectiveinhibitory action, and thus it is expected as a useful antitumor agentin the field of pharmaceuticals.

1. A compound of Formula (I):

wherein: n is 0 or 1; X is O or CH₂; R¹ is H or C₁₋₂ alkyl; R² is H or C₁₋₃ alkyl; R³ and R⁴ are each independently H or C₁₋₂ alkyl, where the alkyl may be substituted with one to three of the same or different substituents selected from R¹⁰; R⁵ is H or OCH₃; R¹⁰ is F or Cl; or a pharmaceutically acceptable salt or ester thereof.
 2. The compound according to claim 1 or a pharmaceutically acceptable salt or ester thereof, wherein R¹ is H or methyl.
 3. The compound according to claim 2 or a pharmaceutically acceptable salt or ester thereof; wherein R² is H or methyl.
 4. The compound according to claim 3 or a pharmaceutically acceptable salt or ester thereof, wherein R³ and R⁴ are each independently H or methyl which may be substituted with one to three of the same or different substituents selected from R¹⁰; and R¹⁰ is F.
 5. The compound according to claim 4 or a pharmaceutically acceptable salt or ester thereof, wherein the compound of Formula (I) is selected from:


6. A compound which is: (a) (2S)-1,1,1-trifluoropropan-2-yl (3aR,6aR)-3a-methoxy-5-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b]pyrrole-1(2H)-carboxylate; (b) (2S)-1,1,1-trifluoropropan-2-yl (4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate; or (c) 1,1,1-trifluoro-2-methylpropan-2-yl (4aR,7aR)-6-[4-methyl-6-(1H-pyrazol-3-ylamino)pyrimidin-2-yl]hexahydropyrrolo[3,4-b][1,4]oxazine-4(4aH)-carboxylate or a pharmaceutically acceptable salt or ester thereof.
 7. A pharmaceutical composition comprising, together with pharmaceutically acceptable carrier or diluent, at least one compound according to claim 1 as active ingredient.
 8. An Aurora A selective inhibitor comprising, together with a pharmaceutically acceptable carrier or diluent, at least one compound according to claim 1 as active ingredient.
 9. An antitumor agent comprising, together with a pharmaceutically acceptable carrier or diluent, at least one compound according to claim 1 as active ingredient. 